Methods for manufacturing betulinic acid

ABSTRACT

The present invention provides a method for preparing betulin-3-acetate including alcoholyzing betulin 3,28-dibenzoate; a process for preparing betulin-3-acetate including: (1) acylating betulin to provide betulin 3,28-dibenzoate and (2) alcoholyzing betulin 3,28-dibenzoate to provide betulin-3-acetate; and a process for preparing betulinic acid including: (1) acylating betulin to provide betulin 3,28-dibenzoate; (2) alcoholyzing betulin 3,28-dibenzoate to provide betulin-3-acetate; (3) oxidizing betulin-3-acetate to provide betulinic aldehyde-3-acetate; (4) oxidizing betulinic aldehyde-3-acetate to provide betulinic acid-3-acetate; and (5) deprotecting betulinic acid-3-acetate to provide betulinic acid.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part application of U.S.application Ser. No. 09/480,406; filed on Jan. 11, 2000.

BACKGROUND OF THE INVENTION

[0002] Betulinic acid is useful as a therapeutic agent. For example,Pisha, E. et al., (1995) J. M. Nature Medicine, 1, 1046-1051 disclosethat betulinic acid has antitumor activity against melanoma, e.g.,MEL-1, MEL-2 and MEL-4. In addition, Fujioka, T. et al., J. Nat. Prod.,(1994) 57, 243-247 discloses that betulinic acid has anti-HIV activityin H9 lymphocytic cells.

[0003] Betulinic acid can be manufactured from betulin, which is presentin large quantities in the outer birch bark of numerous species of birchtrees. For example, a single paper mill in northern Minnesota generatesnearly 30-70 tons of birch bark per day. Approximately 230,000 tons ofbirch bark are generated per year. Outer bark of Betula verrucosa(European commercial birth tree) contains nearly 25% betulin (RainerEkman, 1983, Horzforschung 37, 205-211). The outer bark of Betulapaparifera (commercial birch of northern U.S. and Canada) containsnearly 5-18% betulin (see, U.S. Pat. Ser. No. 09/371298). As such, vastquantities of betulin are available.

[0004] U.S. Pat. No. 5,804,575 issued to Pezzuto et al. discloses afive-step process for the synthesis of betulinic acid from betulin. Dueto the length of time required to carry out this process and the yieldit provides, it is not ideal for the commercial scale (e.g., kilogram)production of betulinic acid. Additionally, the process uses solventsand reagents that are hazardous and expensive, and the disclosedpurification steps are not feasible on a commercial scale.

[0005] The first step in the preparation of betulinic acid frombetulin-3-acetate was described by Ruzichka et al. (Helv. Chim. Acta.,21, 1706-1715 (1938)). The main obstacle for employing this method isthe preparation of starting material (i.e., betulin-3-acetate). Theselectivity of the hydrolysis of betulin-3,28-diacetate with potassiumhydroxide provided about 60% betulin-3-acetate. The use of magnesiumalcoholates (Yao-Chang Xu et al., J. Org. Chem., 61, 9086-9089 (1996))in the selective deprotection of betulin-3,28-diacetate (Yao-Chang Xu etal., J. Org. Chem., 61, 9086-9089 (1996)) has several serious drawbacks.The selectivity of this process is about 81%. Additionally, the cost ofmagnesium alcoholates is fairly high. As such, this method is notattractive for the commercial scale production of betulinic acid.

[0006] Thus, there exists a need for improved methods for preparingbetulinic acid and synthetic precursors thereof. Such improved methodsshould require less time, should provide a higher overall yield, shouldbe more cost effective (i.e., should require less expensive reagents andsolvents) than known procedures, or should satisfy the contemporaryindustrial demands from both safety and environmental points of view.

SUMMARY OF THE INVENTION

[0007] The present invention provides a method for preparing betulinicacid and precursors thereof. The methods of the present inventionrequire less time and require reagents that are less expensive, lesstoxic, or less flammable than known methods for preparing betulinic acidand the precursors thereof.

[0008] The present invention provides a process for preparing a compoundof formula III

[0009] comprising alcoholyzing a compound of formula II

[0010] wherein each R₁ and R₂ are each independently a suitable organicgroup.

[0011] The present invention also provides a process for preparing acompound of formula V

[0012] comprising:

[0013] (1) acylating a compound of formula I

[0014] to provide a compound of formula II

[0015] wherein each R₁ and R₂ are each independently a suitable organicgroup;

[0016] (2) alcoholyzing a compound of formula II to provide a compoundof formula Ill;

[0017] (3) oxidizing the compound of formula III to provide a compoundof formula VI;

[0018] (4) oxidizing the compound of formula VI to provide a compound offormula IV; and

[0019] (5) deprotecting the compound of formula IV to provide thecompound of formula V.

[0020] The invention also provides novel compounds disclosed herein, aswell as methods for their synthesis.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIG. 1 is a schematic illustration of a synthesis of betulinicacid from betulin.

DETAILED DESCRIPTION OF THE INVENTION

[0022] As illustrated in FIG. 1 (scheme 1), Applicant has discovered aprocess for alcoholyzing the bisprotected alcohol (compound II) toprovide the corresponding alcohol (compound III). The alcohol (compoundIII) can be oxidized to the acid (compound IV) and the acid (compoundIV) can be deprotected to betulinic acid (compound V); or the alcohol(compound III) can be oxidized to the aldehyde (compound VI), thealdehyde (compound VI) can be oxidized to the acid (compound IV), andthe acid (compound IV) can be deprotected to provide betulinic acid(compound V); or the alcohol (compound III) can be oxidized to thealdehyde (compound VI), the aldehyde (compound VI) can be deprotected tothe aldehyde (compound VII), and the aldehyde (compound VII) can beoxidized to betulinic acid (compound V).

[0023] The following definitions are used, unless otherwise described:halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl,etc. denote both straight and branched groups; but reference to anindividual radical such as “propyl” embraces only the straight chainradical, a branched chain isomer such as “isopropyl” being specificallyreferred to.

[0024] (C₆-C₁₀)aryl denotes a phenyl radical or an ortho-fused bicycliccarbocyclic radical having about nine to ten ring atoms in which atleast one ring is aromatic.

[0025] Specific and preferred values listed below for radicals,substituents, and ranges, are for illustration only; they do not excludeother defined values or other values within defined ranges for theradicals and substituents.

[0026] Specifically, (C₁-C₁₀)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl,octyl, nonyl or decyl;

[0027] (C₁-C₁₀)alkoxy can be methoxy, ethoxy, propoxy, butoxy,iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, hexyloxy, heptyloxy,octyloxy, nonyloxy, or decyloxy;

[0028] (C₂-C₁₀)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl,1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl,1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl,7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl,6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl,4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, or 9-decenyl;

[0029] (C₂-C₁₀)alkynyl can be ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl,1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl,1-octynyl, 2-octynyl, 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl,7-octynyl, 1-nonylyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 5-nonynyl,6-nonynyl, 7-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 3-decynyl,4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, or 9-decynyl;

[0030] (C₆-C₁₀)aryl can be phenyl, indenyl or naphthyl; and

[0031] (C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl.

[0032] As illustrated in FIG. 1, the alcohol (compound I) can beacylated to provide the bisprotected alcohol (compound II). R₁ and R₂can be any suitable organic group provided R₂C(═O)— can be selectivelyremoved (e.g., alcoholyzed) in the presence of R₁. Suitable organicgroups include (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl,(C₆-C₁₀)aryl, (C₁-C₁₀)alkoxy or (C₆-C₁₀)aryl(C₁-C₁₀)alkyl, wherein anyalkyl, alkoxy, alkenyl, alkynyl, aryl or arylalkyl of R₁ and R₂ can beoptionally substituted with one or more halo, nitro, cyano,trifluoromethyl, hydroxy, SR or NRR, wherein each R is independently Hor (C₁-C₁₀)alkyl. Specifically, R₁ and R₂ can each independently be(C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, or (C₆-C₁₀)aryl. Morespecifically, R₁ and R₂ can independently be (C₁-C₁₀)alkyl (e.g.methyl).

[0033] The acylation can be carried out employing any suitable acylatingreagent (e.g., an acid anhydride, a carboxylic acid, or an acidchloride). Suitable acid anhydrides include acetic anhydride, benzoylanhydride, maleic anhydride, phtalic anhydride, and succinic anhydride.Suitable carboxylic acids include acetic acid and benzoic acid. Suitableacid chlorides include acetyl chloride, pentanoyl chloride and benzoylchloride. In addition, the acylation can be carried out employing anysuitable alkyl acylate (e.g., alkyl acetate). Specifically, theacylating reagent can be acetic anhydride, acetyl chloride or aceticacid.

[0034] The acylation can be carried out at any suitable temperature thatallows for the acylation of both the primary and secondary alcohols.Specifically, the reaction can be carried out at a temperature aboveabout 25° C. or above about 70° C. More specifically, the reaction canbe carried out at a temperature above about 100° C. or above about 118°C.

[0035] The acylation can be carried out in any suitable solvent.Suitable solvents include ethers, esters, acetone, methylene chloride,chloroform, carbon tetrachloride, THF, dicloromethane, pyridine, andaromatic solvents (e.g., benzene, xylenes, toluene,trifluoromethylbenzene, o-chlorotrifluoromethylbenzene,m-chlorotrifluoromethylbenzene, and p-chlorotrifluoromethylbenzene).

[0036] The acylation can be carried out for any suitable length of time.The length of time will vary and depends in part upon the nature andamount of reagents and solvents as well as the temperature of thereaction. Typically, the acylation can proceed for less than about 10hours, less than about 7 hours, or less than about 3 hours.

[0037] Other suitable acylating reagents and reaction conditions areknown in the art, for example see Greene, T. W.; Wutz, P. G. M.,Protecting Groups In Organic Synthesis, second edition, 1991, New York,John Wiley & sons, Inc and Morrison, R. and Boyd, R., Organic Chemistry,672-674, third edition, 1977, Boston, Allyn and Bacon.

[0038] The alcohol (compound I) can be acylated to the bisprotectedalcohol (compound II) employing acetic anhydride/acetic acid (i.e.,Ac₂O/AcOH) in a yield greater than about greater than about 90%, greaterthan about 95%, or greater than about 99%. In addition, the acylationtypically provides compound II (i.e., bis acylated compound) and themono acylated compounds (the compound wherein the primary hydroxyl groupis acylated and the secondary hydroxyl group remains unreacted and thecompound wherein the secondary hydroxyl group is acylated and theprimary hydroxyl group remains unreacted) in a ratio of about 90:10, andpreferably in a ratio of at least about 95:5, or in a ratio of at leastabout 98:2.

[0039] As illustrated in FIG. 1, the bisprotected alcohol (compound II)can be alcoholyzed to provide the alcohol (compound III). As usedherein, “alcoholize,” includes removing a primary acyl group to providea primary hydroxyl group, while a secondary acyl group present on thesame compound is not removed to any appreciable degree. Accordingly, thealcoholysis of a compound containing both a primary acyl group and asecondary acyl group can provide a mixture of compounds (e.g., thecompound wherein the primary acyl group, but not the secondary acylgroup, is alcoholyzed; the compound wherein both the primary andsecondary acyl groups are alcoholyzed; and the compound wherein thesecondary acyl group, but not the primary acyl group, is alcoholyzed).The alcoholysis typically provides the compound wherein the primary acylgroup, but not the secondary acyl group, is alcoholyzed and the compoundwherein both the primary and secondary acyl groups are alcoholyzed, in aratio of about 80:20, and preferably in a ratio of at least about 90:10,or in a ratio of at least about 95:5.

[0040] The alcoholysis can be carried out employing any suitable boronalkoxide (e.g., boron methoxide, boron ethoxide, boron isopropoxide, orboron butoxide). As used herein, “boron alkoxide” is B(O—R)₃ wherein R₃is (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, or (C₂-C₁₀)alkynyl.

[0041] A specific boron alkoxide is boron methoxide B(OMe)₃, boronethoxide B(OEt)₃, boron isopropoxide B(O-iso-propyl)₃, boron n-propoxideB(O-n-propyl)₃, boron sec-butoxide B(O-sec-butyl)₃, or borontert-butoxide B(O-tert-butyl)₃. Specifically, boron alkoxide is boronisopropoxide B(O-iso-propyl)₃, as illustrated in the Examples.

[0042] The alcoholysis can be carried out in any suitable solvent (e.g.,an anhydrous alcohol). Particular alcohols for use in the presentinvention will typically have between about 1 and about 10 carbon atoms;can be cyclic or aliphatic; can be saturated or unsaturated; and can bebranched or straight-chained. Specific alcohols suitable for use in thepresent invention include methanol, ethanol, tert-butanol, andiso-propanol. Specifically, the alcohol is iso-propanol, as illustratedin the Examples. More specifically, from an environmental or recyclingpoint of view, the same alcohol from which the boron alkoxide wasprepared can be employed. For example, if boron iso-propoxide isemployed, isopropanol can be employed as a solvent for alcoholysis.

[0043] The alcoholysis can be carried out at any suitable temperature.Specifically, the alcoholysis can be carried out at a temperature aboveabout 25° C. More specifically, the alcoholysis can be carried out at atemperature above about 50° C. or above about 75° C. (i.e., the boilingtemperature of the solvent).

[0044] The alcoholysis can be carried out for any suitable length oftime, provided the primary acyl group is selectively removed (i.e., theprimary acyl group is removed to a greater degree than the secondaryacyl group). Specifically, the alcoholysis can be carried out for about0.5 to about 4 hours, for about 1 to about 3 hours, or about 1 to about2 hours, as illustrated in the Examples.

[0045] Applicant has discovered that a boron alkoxide in an anhydrousalcohol will selectively cleave (i.e., selectively alcoholize) theprimary acyl group of compound II in the presence of the secondary acylgroup. Specifically, the alcoholysis typically provides compound III(i.e., product) and compound II (i.e., starting material) in a ratio ofabout 80:20, and preferably in a ratio of at least about 90:10, in aratio of at least about 95:5 or in a ratio of at least 98:2. Inaddition, the alcoholysis typically provides compound III and thecompound wherein both the primary acyl group and the secondary acylgroup of compound II are alcoholyzed, in a ratio of about 80:20, andpreferably in a ratio of at least about 90:10, in a ratio at least about95:5, or in a ratio of at least 98:2.

[0046] As illustrated in FIG. 1, the alcohol (compound III) can beoxidized to the aldehyde (compound VI). Applicant has discovered thatthe oxidation can be carried out employing oxalyl chloride (i.e.,(COCl)₂) in dimethyl sulfoxide (i.e., DMSO) in methylene chloride (i.e.,CH₂Cl₂). Specifically, the oxidation typically provides compound VI(i.e., product) and compound III (i.e., starting material) in a ratio ofabout 80:20, and preferably in a ratio of at least about 90:10, or in aratio of at least about 95:5. In addition, the oxidation typicallyprovides compound VI (i.e., product) in a yield of at least about 80%,and preferably at least about 85%, or at least about 95%, based onstarting material (i.e., compound III). Specifically, the oxidationtypically provides compound VI (i.e., product) in a yield of at leastabout 95% if no purification step is necessary.

[0047] Alternatively, the alcohol (compound III) can be oxidized to thealdehyde (compound VI) employing oxygen (i.e., O₂) on palladium acetate(i.e., Pd(OAc)₂), as illustrated in the Examples. Specifically, theoxidation typically provides compound VI (i.e., product) and compoundIII (i.e., starting material) in a ratio of about 80:20, and preferablyin a ratio of at least about 90:10, or in a ratio of at least about95:5. In addition, the oxidation typicaly provides compound VI (i.e.,product) in a yield of about 70%, and typically at least about 80%, orat least about 85%, based on starting material (i.e., compound III).

[0048] The oxidation can be carried out at any suitable temperature,provided the alcohol is oxidized to the aldehyde and the secondary acylgroup remains intact. Specifically, the oxidation can be carried out ata temperature below about −30° C., below about −40° C., or below about−50° C. More specifically, the oxidation can be carried out at atemperature between about −50° C. and about −60° C.

[0049] The oxidation can be carried out for any suitable length of time,provided the alcohol is oxidized to the aldehyde and the secondary acylgroup remains intact. Specifically, the oxidation can be carried out forabout 1 to about 5 hours, for about 1 to about 3 hours, or about 1 toabout 2 hours, as illustrated in the Examples.

[0050] Other reagents suitable for oxidizing an alcohol to an aldehydeare known in the art, for example see Carey, F. & Sundberg, R., AdvancedOrganic Chemistry, 481-490, second edition, Vol. B, Plenum Press, NewYork and London and Morrison, R. and Boyd, R., Organic Chemistiy,520-587, third edition, 1977, Boston, Allyn and Bacon.

[0051] As illustrated in FIG. 1, the aldehyde (compound VI) can beoxidized to the carboxylic acid (compound IV). The carboxylic acid(compound IV) can be isolated as the free acid, or can be isolated as asalt of the carboxylic acid. Specifically, the salt can be apharmaceutically acceptable salt. Applicant has discovered that theoxidation can be carried out employing oxygen and NHPI (or ABIN orTEMPO) in trifluoromethylbenzene, which is commercially available asOXSOL 2000® from Occidental Chemical Corporation (Oxychem). As usedherein, NHPI is N-hydroxyphthalimide. As used herein, “AIBN” is2,2′-Azobisisobutyronitrile and is commercially available from Aldrich(Milwaukee, Wis.) and “TEMPO” is 2,2,6,6-tetramethyl-1-piperidinyloxy,fee radical and is commercially available from Aldrich (Milwaukee,Wis.). Specifically, the oxidation typically provides compound IV (i.e.,product) and compound VI (i.e., starting material) in a ratio of about80:20, and preferably in a ratio of at least about 90:10, or in a ratioof at least about 95:5. In addition, the oxidation typically providescompound IV (i.e., product) in a yield of about 65%, and preferably atleast about 70%, or at least about 80%, based on starting material(i.e., compound VI).

[0052] Alternatively, the aldehyde (compound VI) can be oxidized to thecarboxylic acid (compound IV) employing cobalt(III)acetylacetonate(i.e., Co(AcAce)₃) trifluoromethylbenzene, and oxygen, as illustrated inthe Examples. Specifically, the oxidation typically provides compound IV(i.e., product) and compound VI (i.e., starting material) in a ratio ofabout 80:20, and preferably in a ratio of at least about 90:10, or in aratio of at least about 95:5. In addition, the oxidation typicallyprovides compound IV (i.e., product) in a yield of about 85%, andpreferably at least about 90%, or at least about 95%, based on startingmaterial (i.e., compound VI).

[0053] The oxidation can be carried out at any suitable temperature,provided the aldehyde is oxidized to the carboxylic acid and thesecondary acyl group remains intact. Specifically, the oxidation can becarried out at a temperature between about 30° C. and about 70° C.,between about 40° C. and about 60° C., or between about 45° C. and about50° C.

[0054] The oxidation can be carried out for any suitable length of time,provided the aldehyde is oxidized to the carboxylic acid and thesecondary acyl group remains intact. Specifically, the oxidation can becarried out for about 1 to about 10 hours, for about 4 to about 8 hours,about 5 to about 7 hours, or about 6 hours, as illustrated in theExamples.

[0055] Other suitable oxidative reagents and reaction conditions areknown in the art, for example see Carey, F. & Sundberg, R., AdvancedOrganic Chemistry, 481-490, second edition, Vol. B, Plenum Press, NewYork and London and Morrison, R. and Boyd, R., Organic Chemistry,520-587, third edition, 1977, Boston, Allyn and Bacon.

[0056] As illustrated in FIG. 1, the alcohol (compound III) can beoxidized directly to the carboxylic acid (compound IV). The carboxylicacid (compound IV) can be isolated as the free acid, or can be isolatedas a salt of the carboxylic acid. Specifically, the salt can be apharmaceutically acceptable salt. The oxidation can be carried outemploying any suitable oxidative reagent (see, for example, March,Advanced Organic Chemistry Reactions, Mechanism and Structure, 2nd Ed.,McGraw Hill, 1977, pp. 1107-1108, and references cited therein).Suitable oxidative reagents include: 1)chromium (IV) oxide (i.e., CrO₃)and acetic acid (i.e., HOAc) (J. Am. Chem. Soc., 78, 2255 (1956)); 2)chromium (IV) oxide (i.e., CrO₃) and sulfuric acid (i.e., H₂SO₄) (J. Am.Chem. Soc. 48, 4404 (1983)); 3) potassium permanganate (i.e., KMnO₄)(Tet. Lett., 28, 5263 (1987); J. Am. Chem. Soc., 109, 7280 (1987)); 4)sodium manganate (i.e., NaMnO₄) (Tet. Lett., 22, 1655 (1981)); or 5)palladium chloride (i.e., PdCl₂), potassium carbonate (i.e., K₂CO₃)(Chem. Lett., 1171 (1981)).

[0057] As illustrated in FIG. 1, the acid (compound IV) can bedeprotected to provide betulinic acid (compound V). Betulinic acid(compound V) can be isolated as the free acid, or can be isolated as asalt of betulinic acid (compound V). Specifically, the salt can be apharmaceutically acceptable salt. Accordingly, the secondary acyl groupcan be removed.

[0058] Specifically, the deprotection can be carried out by hydrolysis.The deprotection (e.g., hydrolysis) can be carried out employing anysuitable conditions (e.g., under acidic or basic conditions). Suitableacids include mineral acids (e.g., hydrochloric acid, nitric acid,sulfuric acid, and phosphoric acid). Suitable bases include metalhydroxides and carbonates (e.g., calcium carbonate and potassiumcarbonate). Suitable metal hydroxides include alkaline hydroxides (e.g.,lithium hydroxide, sodium hydroxide and potassium hydroxide) andalkaline earth metal hydroxides (e.g., magnesium hydroxide and calciumhydroxide). Specifically, the hydrolysis can be carried out using sodiumhydroxide in methanol, as illustrated in the Examples.

[0059] The deprotection (e.g., hydrolysis) can be carried out in anysuitable solvent. Suitable solvents include an aqueous alcohol solution(i.e., alcohol and water) wherein alcohol is described hereinabove. Inaddition, the deprotection can be carried out at any suitabletemperature that allows for the deprotection of the secondary acylgroup. Specifically, the deprotection can be carried out at atemperature above about 25° C., above about 50° C. or above about 65° C.More specifically, the deprotection can be carried out at the boilingpoint of the solvent (e.g., if the solvent is methanol and water, thedeprotection can be carried out at a temperature of about 105° C. toabout 120° C.).

[0060] The deprotection can be carried out for any suitable length oftime. Specifically, the deprotection can be carried out for about 1 toabout 24 hours, for about 1 to about 10 hours, or for about 1 to about 3hours, as illustrated in the Examples.

[0061] Other suitable deprotection (e.g., hydrolysis) conditions areknown in the art, for example see Carey, F. & Sundberg, R., AdvancedOrganic Chemistry, 481-490, second edition, Vol. B, Plenum Press, NewYork and London and Morrison, R. and Boyd, R., Organic Chemistry,675-682, third edition, 1977, Boston, Allyn and Bacon.

[0062] Applicant has discovered a one-pot, synthetically viable, andcost-efficient method to prepare the alcohol (compound III) from betulin(compound I). The one-pot process is faster and less expensive thanknown methods. The monoprotected alcohol (compound III) can be oxidizedto the acid (compound IV) which can be deprotected to betulinic acid(compound V). Alternatively, the monoprotected alcohol (compound III)can be oxidized to the aldehyde (compound VI), and the aldehyde(compound VI) can be oxidized to the acid (compound IV), and deprotectedto betulinic acid (compound V). All of the procedures described hereinabove and illustrated in the Examples herein below can be scaled up toindustrial scale (i.e., kilogram).

[0063] The present invention will be described by the followingexamples. The examples are for illustration purposes and do nototherwise limit the invention.

EXAMPLES

[0064] Betulin was obtained from outer birch bark employing the methodsas disclosed in co-pending U.S. Application No. 09/371,298; Elkman, R.,(1983) Holzforsch, 37, 205; Ohara, S., et al., (1986) Mokuza Gakkaishi,32, 266; and Eckerman, C., (1985) Paperi ja Puu, No. 3, 100.

Example 1 Betulin-3-Acetate (III)

[0065]

[0066] Acetic anhydride (85 ml) and acetic acid (800 ml) are introducedinto round bottom flask (3 L). Betulin (I) (100 g, 0.2259 mol) is addedto the stiffed solution and refluxed for 3 hours. The reaction mixtureis allowed to cool to 50° C. and acetic acid was evaporated underreduced pressure (25-30 mm Hg). Cream-white crystals of intermediatebetulin-3,28-diacetate (II) are obtained after storage in vacuo.

[0067] Isopropanol (i.e., i-PrOH) (2.5 L) and powdered boroniso-propoxide (i.e., B(O-i-Pr)₃) (100 g, 0.223 mol) are added to theflask and the mixture is refluxed for 1.5 hours. Isopropyl alcohol isthen removed under reduced pressure (100 mm Hg) at 30-33° C. Theresulting white-orange crystals are dissolved in dichloromethane (1 L)and water (60 ml) is added to the solution. After stirring (10-15minutes) the precipitated material is filtered, extracted withdichlorometbane (4×200 ml) and dried over sodium sulfate (10 g). Solventevaporation provides white crystals. Crystallization fromtrifluoromethylbenzene yields betulin 3-acetate (III).

Example 2 Betulinic Aldehvde-3-Acetate (VI)

[0068]

[0069] Oxalyl chloride (15.72 g, 0.1237 mol) in methylene chloride(i.e., CH₂Cl₂) (500 ml) is placed in a round bottom two-neck flask (2L). After cooling to −50 to −60° C. (dry ice bath) and with efficientstirring is added dimethylsulfoxide (i.e., DMSO) (12.87 g, 0.165 mol) inmethylene chloride (500 ml) dropwise over 5 to 10 minutes. The mixtureis stirred for an additional 5 to 10 minutes (until gas evolutionstops). Betulin-3-acetate (III) (powdered) (0.0825 mol, 40 g) is thenadded and allowed to stand for 45 minutes. After triethylamine (41.67 g,0.4126 mol) is added, the cooling bath is removed and temperatureallowed to increase to 10° C. Cold water (200 ml) is added and themixture is extracted with methylene chloride (3×100 ml). The combinedorganic extracts are washed with water (5×100 ml), 5% HCl (2×100 ml) andbrine (2×100 ml). After drying over sodium sulfate (10 g), evaporationof the solvent gives crude compound, which after crystallization fromhexane gives white crystalline product, betulinic aldehyde-3-acetate(VI).

Example 3 Betulinic Aldehyde-3-Acetate (VI)

[0070]

[0071] Oxalyl chloride (7.86 g, 62 mmol) in trifluoromethylbenzene (250ml) is placed in a round bottom two-neck flask (2 L). After cooling to−30 to −35° C. (i-PrOH-dry ice bath) and with efficient stirring isadded dimethylsulfoxide (6.44 g, 83 mmol) in trifluoromethylbenzene (250ml) drop-wise over 5 to 10 minutes. The mixture is stirred for anadditional 5-10 minutes (until gas evolution stops). Powdered3-O-acetyl-betulin (III) (40 g, 41 mmol) is then added. The resultingmixture is allowed to stand for 45 minutes. After triethylamine (41.67g, 206 mmol) is added, the cooling bath is removed and the temperatureis allowed to increase to 10° C. Cold water (100 ml) is added and themixture is extracted with trifluoromethylbenzene (3×50 ml). The combinedorganic extracts are washed with water (5×50 ml), 5% HCl (2×50 ml) withbrine (2×50 ml). After drying over sodium sulfate (5 g), evaporation ofthe solvent gives crude compound, which after crystallization fromhexane gives white crystalline product, betulinic aldehyde-3-acetate(VI).

Example 4 Betulinic Aldehyde-3-Acetate (VI)

[0072]

[0073] Palladium acetate (i.e., Pd(OAc)₂) (112 mg, 0.5 mmol) intrifluoromethylbenzene (500 ml) is introduced into a round bottom twoneck flask (IL). Pyridine (158 mg, 2 mmol) and MS3A (i.e., molecularsieves, 3 Å) (5 g) are then added. The mixture is heated to 80 to 85° C.and oxygen was purged for 10 minutes. Betulin-3-acetate (III) (powdered)(4.84 g, 10 mmol) is added and oxygen is passed through the system at 80to 85° C. for 1.5 hours. The mixture is filtered through silica gel (25g, 230-400 mesh) to remove the catalyst. Evaporation of the solvent invacuo gives crude product, which after crystallization from hexaneyields white crystals of betulinic aldehyde-3-acetate (VI).

Example 5 Betulinic Acid-3-Acetate (IV)

[0074]

[0075] Betulinic aldehyde-3-acetate (VI) (3 g, 62.15 mmol) is dissolvedin trifluoromethylbenzene (120 ml) and then was placed into a two-neckround bottom flask (250 ml). Methanol (1.2 ml) and NHPI (53 mg, 0.32mmol) is added to the solution. Oxygen is bubbled through the solutionat 15 to 20° C. for 2 hours. The reaction mixture is poured into coldwater (50 ml) and extracted with trifluoromethylbenzene (2×20 ml). Thecombined organic fractions are washed with 1% aqueous solution of sodiumbicarbonate until the water layer is colorless (4×30 ml). Thetrifluoromethylbenzene solution is dried over sodium sulfate (0.5 g) andthe solvent evaporated in vacuo at 27 to 30° C. to give the crudeproduct.

[0076] Crystallization from MeOH-hexane gives white crystals ofbetulinic acid-3-acetate (IV):

[0077] Alternatively 3-acetyl-betulinic acid (IV) can be separatedthrough the methyl ester by purification on silica (230-400 mesh,hexane: ether=3:1).

Example 6 Betulinic Acid (V)

[0078]

[0079] Betulinic aldehyde-3-acetate (VI) (1.455 g, 30.14 nunol) intrifluoromethylbenzene (150 ml) is placed into a round bottom two-neckflask (300 ml). Cobalt (III) acetylacetonate (75 mg, 0.205 mmol) intrifluoromethylbenzene (4 ml) is added to the solution and oxygen wasbubbled through the mixture at 60-65° C. for 1 hour.Trifluoromethylbenzene is evaporated under reduced pressure 20 mm Hg, at40° C.) and sodium hydroxide (0.82 g, 0.0205 mol) in MeOH (100 ml) isadded and the mixture boiled for 1.5 hours. Methanol is evaporated underreduced pressure (130 mm Hg, 40° C.), water (150 ml) is added and themixture is acidified with 15% hydrocloric acid (pH is about 4). Theprecipitate is filtered and washed with water (3×100 ml).Crystallization from MeOH gives white crystals of betulinic acid (V).

Example 7 Betulinic Acid (v)

[0080]

[0081] Betulinic acid-3-acetate (IV) (0.5 g, 1.003 mmol) is refluxed inmethanol (50 ml) containing sodium hydroxide (0.3 g, 7.5 mmol) for 1hour. The reaction mixture is then diluted with 100 ml of water andfiltered to provide a white residue. The white residue is crystallized(methanol-hexanes) to provide white crystals of betulinic acid (V).

Example 8 Betulin 3,28-Dibenzoate (II)

[0082]

[0083] Betulin (I) (2 g, 4.52 mmol), benzoylchloride (1.40 g, 9.96 mmol)and pyridine (25 mL) are introduced into round bottom flask (50 mL). Themixture is maintained at 50-60° C. for 24 hours. The solution is pouredinto dichloromethane (100 mL) and washed with water (2×50 mL), 1% HCl(50 mL) and water until a neutral reaction of universal indicator paperis observed (i.e., pH=7). Chromatography on a silica column (Ether:Hexane=25:75) yields white crystals of betulin 3,28-dibenzoate (II):

Example 9 Betulin 3-O-Benzoate (III)

[0084]

[0085] A stirred solution of betulin dibenzoate (II) (1 g, 1.54 mmol) inisopropanol (25 ml) is maintained in a round bottom flask (50 ml).Powdered boron isopropoxide (i.e., B(OiPr)₃) (1.56 g, 7.7 mmol) is addedand the mixture is refluxed for twenty-four hours. Isopropyl alcohol isdistilled under reduced pressure (100 mm Hg) at 30-33° C. The residue isdissolved in dichloromethane (50 ml) and water (2.07 ml) is added to thesolution. After stirring (15 minutes) the resulting precipitate isfiltered and washed with dichloromethane (4×10 ml) and dried over sodiumsulfate (1 g). Solvent evaporation gives white crystals ofbetulin-3-benzoate (III).

[0086] All publications, patents, and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications can be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A process for preparing a compound of formula III

comprising: (1) acylating a compound of formula I

to provide a corresponding compound of formula II

wherein R₁ and R₂ are each independently any suitable organic group; and(2) alcoholyzing a compound of formula II to provide a correspondingcompound of formula III.
 2. The process of claim 1 wherein the acylatingcomprises heating to reflux in acetic acid and acetic anhydride forabout 2 hours to about 5 hours.
 3. The process of claim 1 wherein theacylating comprises heating in pyridine and benzoyl chloride at about50° C. to about 60° C. for about 20 hours to about 30 hours.
 4. Theprocess of claim 1 wherein the alcoholyzing comprises heating in thepresence of a boron alkoxide and an anhydrous alcohol.
 5. The process ofclaim 1 wherein R₁ and R₂ are each (C₁-C₁₀)alkyl.
 6. The process ofclaim 5 wherein (C₁-C₁₀)alkyl is methyl.
 7. The process of claim 6wherein the boron alkoxide is boron isopropoxide.
 8. The process ofclaim 1 wherein the alcohol is isopropanol.
 9. The process of claim 1further comprising oxidizing the compound of formula III to provide acompound of formula VI


10. The process of claim 9 further comprising oxidizing the compound offormula VI to provide a compound of formula IV


11. The process of claim 10 further comprising deprotecting the compoundof formula IV to provide a compound of formula V


12. A process for preparing the compound of formula V

comprising: (1) acylating a compound of formula I

to provide a corresponding compound of formula II

wherein R₁ and R₂ are each independently any suitable organic group; (2)alcoholyzing a compound of formula II to provide a correspondingcompound of formula III;

(3) oxidizing the compound of formula III to provide a correspondingcompound of formula VI;

(4) oxidizing the compound of formula VI to provide a compound offormula IV; and

(5) deprotecting the compound of formula IV to provide the compound offormula V.


13. The process of claim 12 wherein R₁ and R₂ are each methyl.
 14. Theprocess of claim 12 wherein the alcoholyzing comprises heating thecompound of formula II in the presence of a boron alkoxide and ananhydrous alcohol.
 15. The process of claim 14 wherein the boronalkoxide is boron isopropoxide.
 16. The process of claim 14 wherein thealcohol is isopropanol.
 17. The process of claim 12 wherein theacylating comprises heating to reflux in acetic acid and aceticanhydride for about 2 hours to about 5 hours.
 18. The process of claim12 wherein the acylating comprises heating in pyridine and benzoylchloride at about 50° C. to about 60° C. for about 20 hours to about 30hours.
 19. The process of claim 12 wherein the oxidizing of compound IIIto compound VI comprises palladium acetate, molecular sieves, and oxygenin trifluoromethylbenzene and pyridine at about 80° C. to about 85° C.for about 0.5 hour to about 4 hours.
 20. The process of claim 12 whereinthe oxidizing of compound VI to compound IV comprises oxygen and Cobalt(III) acetylacetonate in trifluoromethylbenzene at 60-65° C. for about0.5 hour to about 2 hours.
 21. The process of claim 12 wherein thedeprotecting comprises heating to reflux in methanol, water and sodiumhydroxide.
 22. A process for preparing a compound of formula III

comprising: alcoholyzing a corresponding compound of formula II

wherein R₁ and R₂ are each independently any suitable organic group; toprovide the formula III.